Content uploaded by Pannawat Chaiyawatthanananthn
Author content
All content in this area was uploaded by Pannawat Chaiyawatthanananthn on May 16, 2021
Content may be subject to copyright.
S192 J Med Assoc Thai Vol. 100 Suppl. 5 2017
Determination of Free Radical Protective Activity from
Hydrogen Peroxide, Antioxidant and Melanogenesis
Stimulating Activities of Eclipta prostrate Linn. and
Sapindus rarak DC. for Hair Dye Product
Yanisa Nakeenopakun BATM*,
Pannawat Chaiyawatthanananth PhD**,***, Arunporn Itharat PhD**,***
* Student of Master of Science (Applied Thai Traditional Medicine), Faculty of Medicine, Thammasat University,
Pathumthani, Thailand
** Department of Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University, Pathumthani, Thailand
*** Center of Excellence on Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University,
Pathumthani, Thailand
Background: Thai medicinal plant namely Eclipta prostrate Linn. (EP) and Sapindus rarak DC. (SR) are reported to
improve hair growth and hair color. Theories of grey hair occurred from the gradual loss of pigmentation. Intrinsic factors
are age, genetic, stress and extrinsic factor such as chemical that involved melanogenesis process and oxidative mechanism.
Moreover, hydrogen peroxide (H2O2) is known as an oxidant which can destroy melanocytes cell. On the other hand, if the
herb extracts can increase melanocytes cell and high antioxidant, they will protect melanocytes cell.
Objective: To investigate free radical protective activity from hydrogen peroxide, antioxidant and melanogenesis stimulating
activities of Eclipta prostrate Linn. and Sapindus rarak DC.
Material and Method: The leaves of EP and the pericarps of SR were maceration in 95% ethanol (EPE95 and SRE95), 50%
ethanol (EPE50 and SRE50), and were decoction with water (EPA and SRA). All extracts were tested antioxidant activity by
DPPH radical scavenging assay and ABTS radical cation decolorization assay. The effect of extracts on melanocytes
proliferation in mouse melanoma cell (B16F10) and in preventing cells from H2O2 by MTT assay were also investigated.
Results: The results showed that EPA had the highest DPPH radical ion inhibition with the EC50 value of 15.00
μ
g/ml. For
ABTS assay, EPE50 and EPA showed the highest inhibitory effect on ABTs radical assay (EC50 = 54.24 and 30.28
μ
g/ml,
respectively). For melanocytes proliferation assay, EPE95 showed the highest stimulating effect on melanocyte proliferation
at the concentration of 50
μ
g/ml. Moreover, the SRA showed high activity on the effect of herbs in preventing cells from H2O2
at the concentration of 10 and 1
μ
g/ml.
Conclusion: These results can be concluded that SRA showed the effect of prevention of cell from H2O2 and EPE95 showed
melanocytes proliferation stimulating effects and EPA showed high antioxidant activities. Thus, these extracts should be
continuously developed as a hair product.
Keywords: Antioxidant, Hydrogen peroxide, Melanocyte proliferation, Grey hair, Eclipta prostrata Linn., Sapindus rarak
DC.
J Med Assoc Thai 2017; 100 (Suppl. 5): S192-S199
Full text. e-Journal: http://www.jmatonline.com
Correspondence to:
Itharat A, Department of Applied Thai Traditional Medicine,
Faculty of Medicine, Thammasat University, Rangsit,
Pathumthani 12121, Thailand.
Phone: +66-2-9269749, Fax: +66-2-9269705
E-mail: iarunporn@yahoo.com
Nowadays, Thailand is an aging society. The
increasing number of hair graying results in decreasing
self-confident, beauty and older than actual age. All
hair has pigment cells called melanocytes. These
melanocytes produce a pigment called melanin, more
specifically, the chemicals eumelanin and pheomelanin.
Hair with more eumelanin will be darker, and hair with
more pheomelanin will have a red/orange/yellow tint.
Hair with less of either will be lighter(1). These
melanocytes also pass this pigment to cells called
keratinocytes (which produce hair’s primary component
and the protein keratin). When the keratinocyte cells
die, they keep the melanin and gives hair its color. As
we get older, the melanocytes get less active, thus
producing of pigment is reduced and making hair
lighter. Eventually, all the melanocyte cells die, and there
J Med Assoc Thai Vol. 100 Suppl. 5 2017 S193
are none left to produce any color(2). However, stressors
induce alterations in pigment-producing, antioxidative
enzymes, cofactors, the production of endogenous
antioxidants and repair enzymes as well as growth
factors. This reaction results in a breakdown of the hair
follicle melanocyte redox-capacity and subsequent
deleterious oxidative stress damage(3).
Hydrogen peroxide is the most commonly
used as an oxidizing agent. The ideal herbal product
was needed in the areas of color saturation, color
development without causing hair damage and
reducing skin irritation. However, the oxidizing of the
oxidative coloring agents which are color from herbs
cannot solubilize and decolorize the colored melanin
component in the hair. Then hydrogen peroxide can be
mixed with herbs to bleach and penetrate the hair shaft
and removes the natural pigment of the hair(4).
In the past, the use of local herbs for hair care
has been continuously use for a long time such as
false-daisy white head (Eclipta prostrata Linn.). It is a
local herb in Thailand, and it can change hair color
from white to black and prevent premature gray hair(5).
In Thai traditional medicine has used juice from fruits
of Sapindus rarak DC. to inhibit the growth of fungi
that cause ringworm, tinea capitis and dandruff(6).
However, there is no report about activity on
melanocyte cells and prevention from H2O2. Thus, the
objective of this research are to investigate antioxidant,
melanocytes proliferation stimulating and cell protection
activities of Eclipta prostrata Linn. (EP) and Sapindus
rarak DC. (SR) extracts.
Material and Method
Chemicals and reagents
Dimethyl sulfoxide [(CH3) 2SO)] (DMSO) was
purchased from RCI Lab scan, Thailand. Hydrochloric
acid (HCl) and Sodium hydroxide (NaOH) were
purchased from Univar, Australia. Dulbecco’s modified
Eagle’s medium (DMEM), Penicillin-Streptomycin
(P/S), Trypan blue stain 0.4% and Trypsin-EDTA were
purchased from Gibco, USA. Phosphate buffered saline
(PBS) was purchased from Amresco, USA. Sodium
bicarbonate (NaHCO3) was purchased from BHD,
England. 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2’-
Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid
(ABTS), 5-Diphenyltetrazolium Bromide (MTT),
Hydrogen peroxide 30% (H2O2) from Qrec, Newzealand,
UV-vis spectrophotometer, microplate reader.
Plant materials
The parts of plants of Eclipta prostrata Linn.
(EP) and Sapindus rarak DC. (SR) were collected from
Pathumthani, Thailand in 2014, voucher specimens
shown in (Table 1). The voucher specimens were
verified at the herbarium of Southern Center for Thai
Medicinal Plants at Faculty of Pharmaceutical Sciences,
Prince of Songkla University, Songkhla, Thailand.
Preparation of crude extracts
Plant materials were cleaned with water,
sliced into small pieces and dried in a hot air oven at
temperature 50°C and its ingredients herbs (200 grams
of each) were macerated with 95% ethanol and
were macerated with 50% ethanol (100 grams of each)
for 3 days, filtered using Whatman No.1 filter paper
and concentrated to dryness by evaporator (Rotavapor
R-205, Germany). The filtrate was pooled and dried
using an evaporator. Each herb (100 grams of each)
was boiled with water (decoction) for 15 minutes in 3
times and filtered through a Whatman No. 1 filter paper.
This residue of aquous extract was repeated twice and
the filtrate was dried by lyophillizer. Percentage yields
of all extracts were calculated by using the following
equation.
Antioxidant activities
2,2-Diphenyl-1-picrylhydrazyl (DPPH)
assay(7)
The antioxidant activity was determined using
2,2-diphenyl-1-picrylhydrazyl (DPPH) as described by
Yamasaki et al 1994(7). A sample for testing was dissolved
in absolute ethanol or distilled water in various
concentrations. 100 μl of extracts were transferred into
a 96-well microplate. Then 100 μl of 6x10-5 M DPPH (in
absolute ethanol) were added to each well. A portion of
the sample was mixed with an equal volume of 6x10-5 M
DPPH. After incubation for 30 min in the dark at room
temperature, the absorbance was measured at 520 nm.
BHT was used as a positive control. The concentration
of antioxidant needed to decrease the initial DPPH
concentration (EC50) by 50% is a parameter widely used
to measure the antioxidant activity. The scavenging
activity was calculated as percentage inhibition in the
formulae below: Inhibition % = [(A Control-A Sample)/
A Control]x100] Effective concentration of sample
required to scavenge DPPH radical by 50% (EC50) was
obtained by linear regression analysis of the dose-
response curve of % inhibition versus concentration,
% yield = x 100
Weight of all extracts (g)
Weight of herb for extracting (g)
S194 J Med Assoc Thai Vol. 100 Suppl. 5 2017
and EC50 is calculated using Prism program. All
determinations were carried out in triplicate.
ABTS radical scavenging assay(8)
Total antioxidant capacity of the extracts
was tested according to ABTS•+ solution which was
produced by reacting 7 mM ABTS•+ stock solution in
distilled water with 2.45 mM potassium persulfate. The
mixture was allowed to stand in the dark at room
temperature for 12 to 16 h. The ABTS•+ solution was
diluted with distilled water to get the absorbance of
0.68 to 0.72 at 734 nm before use. The assay was
performed in 96-well microplates by mixing ABTS•+
solution with 10 μl of the extracts or standard Trolox.
The reaction was carried out for 6 min, and then the
absorbance was measured at 734 nm using a microplate
reader (PowerWave XS, BioTek). The scavenging
activity of the extracts against ABTS•+ was expressed
as EC50 (μg/mL) and Trolox equivalent antioxidant
capacity (TEAC) (mg Trolox equivalents/g extract).
Melanin proliferation by MTT assay
Cell culture(9)
B16F10 murine melanoma cell (ATCC® CRL-
6475TM) were cultured in DMEM containing 10% fetal
bovine serum, 100 U/ml penicillin and 0.1 mg/ml
streptomycin at 37°C in a humidified 95% air 5% CO2
atmosphere.
Melanin proliferation by MTT assay(10)
Melanin proliferation was determined after
treatment with the tested compounds using 3- (4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyltetra-zolium bromide
(MTT; Sigma Chemical Co., St. Louis, USA) which
measures mitochondrial activity in living cells as
described previously. Briefly, B16F10 cells were cultured
at 3,000 cells/well in a 96-well plate. After 24 h, cells
were then treated with 95% or 50% ethanolic or aqueous
extracts at 100 - 1 μg/ml. 100 μl sample solution was
added and mixed where 2% DMSO solution was used
as control solvent. Cells in a 96-well plate were incubated
in the CO2 incubator for 24 hours. Medium was removed
and added 100 μl fresh media. Then incubated with
MTT solution (1 mg/mL) 15 μl for 4 h at 37°C. The
medium was removed and DMSO was added to dissolve
the formazan product in these cells. Absorbance was
measured at 570 nm using a microplate reader and
Proliferation Index were calculated from this equation.
Botanical name % yield Code DPPH assay Code ABTs assay
EC50 + SEM (μg/ml) EC50 + SEM (μg/ml)
95% 50% Aqueous 95% 50% Aqueous 95% 50% Aqueous
ethanol ethanol ethanol ethanol ethanol ethanol
Eclipta prostrata 7.825% 27.70% 23.30% EP 73.46+0.57 >100 15.93+1.79 EP >100 54.24+3.02 30.28+3.77
Linn: Compositae
Sapindus rarak 13.52% 31.79% 21.40% SR >100 >100 >100 SR >100 >100 >100
DC: Sapindaceae BHT 13.40+0.27 13.40+0.27 13.40+0.27 Trolox 5.709+0.501 5.709+0.501 5.709+0.501
(positive) (positive)
Table 1. The percentage of yield of extracts from EP and SR by 95%, 50% ethanolic and aqueous extraction, 50% effective concentration (EC50) of 95%, 50% ethanolic and
aqueous extracts by DPPH assay and ABTs assay (n = 3)
% inhibition = x100
Abs control - Abs sample
Abs control
J Med Assoc Thai Vol. 100 Suppl. 5 2017 S195
Preventing cell for hydrogen peroxide in
mouse melanoma cells (B16F10) by colorimetric
MTT assay(11,12)
Effect of herbs in preventing cell for hydrogen
peroxide with the tested compounds using 3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyltetra-zolium bromide
(MTT; Sigma Chemical Co., St. Louis, USA) which
measures mitochondrial activity in living cells as
described previously(11). Briefly, B16F10 cells were
cultured at 3,000 cells/well in a 96-well plate. After 24 h,
cells were then treated with 95% or 50% ethanolic or
aqueous extracts at 100 to 1 μg/ml. Then 50 micromolar
of H2O2 was added (the concentration which can make
B16F10 cells die in 50%). 100 μl sample solution was
added and mixed where 2% DMSO solution and 50
micromolar of H2O2 were mixed and used as control
solvent. Cells in the 96-well plate were incubated in the
CO2 incubator for 24 hours. Medium was removed from
96 well plate and 100 μl fresh media was added. Then
incubated with MTT solution (1 mg/mL) 15 μl for 4 h at
37°C. The medium was removed, and DMSO was added
to dissolve the formazan product in these cells.
Absorbance was measured at 570 nm using a microplate
reader and Proliferation index were calculated from this
equation. Calculate for % proliferation index.
Statistical analysis
Data are expressed as mean + SEM (n = 3).
IC50 and EC50 values were calculated using GraphPad
Prism software (version 4.03). Student t-test and one-
way ANOVA followed by Duncan’s or Dunnett’s T3
was used to compare all groups. Statistical analysis
was performed using SPSS for Windows (version 16.0).
Statistical significance was set at a level of p<0.05. For
correlation analysis, Pearson’s correlation coefficient
(r) with p-value between antioxidant activities and
contents was identified. The p<0.05 was considered
statistically significant.
Results The percentage of yields (w/w) of the 95%
ethanolic extracts, 50% ethanolic extracts and aqueous
extracts of EP and SR were shown in Table 1. They
were tested antioxidant activity which based on
chemical methods. All extracts showed the results of
P.I = x100
Mean of OD sample
Mean OD of control
P.I = x100
Mean of OD sample
Mean OD of control
DPPH assay and ABTS assay with their particular EC50
value (Table 1). The results showed that EPA showed
the highest inhibitory DPPH radical ion with the EC50
value of 15.00 μg/ml. For ABTS assay, EPE50 and EPA
showed the highest inhibitory effect on ABTs radical
assay (EC50 = 54.24 and 30.28 μg/ml, respectively). From
melanocytes proliferation assay, the proliferation index
of the 95% ethanolic extracts, 50% ethanolic extracts,
and aqueous extracts were shown in Fig. 1. The EPE95
showed the highest stimulating effect on melanocyte
proliferation at the concentration of 50 μg/ml. The
Proliferation index in preventing cell from hydrogen
peroxide of 95%, 50% ethanolic and aqueous extracts
from EP and SR (Fig. 2). The SRA showed high activity
on preventing cells from H2O2 at the concentration of
10 and 1 μg/ml, respectively.
Discussion
In the present study, EP can increase melanin
proliferation and SR as a plant which can inhibit the
growth of fungi that cause ringworm, tinea capitis and
dandruff can protect cell melanocytes from hydrogen
peroxide. The results of the EP water extract related
with the previous report which also showed high
antioxidant , however the antioxidant activity by DPPH
assay of the EP water extract showed higher than the
previous study (IC50 values = 15.93 and 230 μg/ml
respectively)(13). The different results depend on source
of growing, age of plant which make the active
compound difference. The results of antioxidant testing
by DPPH and ABTS assay of EP extract relate in the
same way, the water extract on both antioxidant assay
showed higher effective than ethanolic extract
respectively. For EP 95% ethanolic extract which was
tested by DPPH, showed higher antioxidant than EP
50% ethanolic extract. In the opposite result, EP 95%
ethanolic extract which was tested by ABTS, showed
less antioxidant than EP 50% ethanolic extract. The
results depend on polarity of extract especially ABTS
assay, the polar extraction showed effective than
nonpolar extract. The previous study of the EP 95%
ethanolic extract exhibited high antioxidant activities,
with IC50 = 41.8 mg/ml(14). Our results indicated that
EPE50 and EPA showed the highest inhibitory effect
on ABTs radical assay (EC50 = 54.24 and 30.28 μg/ml,
respectively) but EPE95% had no activity. Thus this
result is opposite the previous study.
The proliferation index of the 95% ethanolic
(EPE95) showed the highest stimulating effect on
melanocyte proliferation at the concentration of 50 μg/
ml which related with the previous study which reported
S196 J Med Assoc Thai Vol. 100 Suppl. 5 2017
Fig. 1 Proliferation index in melanocytes proliferation assay of 95%, 50% ethanolic and aqueous extracts of EP and SR
by MTT assay (n = 3).
Fig. 2 Proliferation index in preventing cell from hydrogen peroxide of 95%, 50% ethanolic and aqueous extracts of EP
and SR (concentration of Hydrogen peroxide to kill 50% melanocyte cells is 0.05 M or 50 micromolar) by MTT
assay (n = 3).
that the methanolic extract of the EP has also efficacy
in promoting hair growth. An extract at dose of 3.2 mg/
15 cm2 showed 87.5%(15). However, this study is the
first report of EP 95% ethanolic extract which was study
in cells. For SR results, it was concluded that all extract
of SR had no antioxidant by both antioxidant assay.
The high concentration of all SR extract can kill
melanocyte cells except SRA at the concentration of 10
and 1 μg/ml can stimulate cell growth and also showed
high proliferate activity when added H2O2. The results
can be concluded that the water extract of SR showed
the highest protecting cells from H2O2 at the
J Med Assoc Thai Vol. 100 Suppl. 5 2017 S197
concentration of 10 and 1 μg/ml, respectively.
This finding indicates that SRA protects cell
from H2O2, EPE95 stimulates melanocytes proliferation
and EPA has high antioxidant activities. Thus, these
extracts should be continuously developed as the
hair product. However, the effect of SRA and EPA on
anti-tyrosinase activity should be the best tested for
strong support data on melanin production.
Conclusion
To our knowledge, these results can be
concluded that SRA protects cell from H2O2, EPE95
stimulates melanocytes proliferation and EPA has high
antioxidant activities. Thus, these extracts should be
continuously developed as the hair product.
What is already known on this topic?
Thai traditional herbs used Eclipta prostrate
Linn. (EP) for dying hair and preventing premature
gray hair. For EP, the aqueous extract of EP showed
antioxidant activities and the methanol extracts of EP
showed efficacy for promoting hair growth. For
Sapindus rarak DC. (SR), the concentration 1, 10, 50,
100 and 200 ppm of SR pulp showed antioxidant
activities and it could inhibit the growth of fungi which
was caused ringworm, tenia capitis and dandruff.
However, SR had no melanin proliferation. In the end,
there had no research in EP and SR about the effect in
preventing cell for hydrogen peroxide activity.
What this study adds?
The present study showed that the aqueous
extract of Eclipta prostrate Linn. (EP) was the highest
antioxidant activities but all extracts of Sapindus rarak
DC. (SR) had no antioxidant activities. In addition, the
95% ethanolic extract of EP in 50 μg/ml concentration
showed the highest melanin proliferation assay.
However, the aqueous of SR in 10 μg/ml concentration
showed the highest effect of herbs in preventing cell
from hydrogen peroxide assay.
Acknowledgements
This work was supported by the National
Research University Project of Thailand Office of
Higher Education Commission and Center of
Excellence on Applied Thai Traditional Medicine
Research (CEATMR). Faculty of medicine, Thammasat
University.
Potential conflicts of interest
None.
References
1. Arck PC, Overall R, Spatz K, Liezman C, Handjiski
B, Klapp BF, et al. Towards a “free radical theory
of graying”: melanocyte apoptosis in the aging
human hair follicle is an indicator of oxidative stress
induced tissue damage. FASEB J 2006; 20: 1567-9.
2. Wood JM, Decker H, Hartmann H, Chavan B, Rokos
H, Spencer JD, et al. Senile hair graying: H2O2-
mediated oxidative stress affects human hair color
by blunting methionine sulfoxide repair. FASEB J
2009; 23: 2065-75.
3. Tobin DJ, Paus R. Graying: gerontobiology of the
hair follicle pigmentary unit. Exp Gerontol 2001;
36: 29-54.
4. Packianathan N, Karumbayaram S. Formulation
and evaluation of herbal hair dye: an ecofriendly
process. J Pharm Sci Res 2010; 2: 648-56.
5. Dalal S, Kataria SK, Sastry K, Rana S.
Phytochemical screening of methanolic extract and
antibacterial activity of active principles of
hepatoprotective herb, Ecliptaalba. Ethnobotanical
Leaflets 2010; 14: 248-58.
6. Rahmadina A. Uji Sitotoksisitas Rebusan Buah
Lerak (Sapindus rarak DC) Terhadap Sel BHK-21
Menggunakan Esei MTT [thesis]. Surabaya:
Universitas Airlangga; 2011.
7. Yamasaki K, Hashimoto A, Kokusenya Y, Miyamoto
T, Sato T. Electrochemical method for estimating
the antioxidative effects of methanol extracts of
crude drugs. Chem Pharm Bull (Tokyo) 1994; 42:
1663-5.
8. Re R, Pellegrini N, Proteggente A, Pannala A, Yang
M, Rice-Evans C. Antioxidant activity applying an
improved ABTS radical cation decolorization
assay. Free Radic Biol Med 1999; 26: 1231-7.
9. Jung GD, Yang JY, Song ES, Par JW. Stimulation of
melanogenesis by glycyrrhizin in B16 melanoma
cells. Exp Mol Med 2001; 33: 131-5.
10. Itoh T, Furuichi Y. Hot-water extracts from adzuki
beans (Vigna angularis) stimulate not only
melanogenesis in cultured mouse B16 melanoma
cells but also pigmentation of hair color in C3H
mice. Biosci Biotechnol Biochem 2005; 69: 873-82.
11. Jitsanong T, Khanobdee K, Piyachaturawat P,
Wongprasert K. Diarylheptanoid 7-(3,4 dihydroxy-
phenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene from
Curcuma comosa Roxb. protects retinal pigment
epithelial cells against oxidative stress-induced cell
death. Toxicol In Vitro 2011; 25: 167-76.
12. Kim KT, Yoo KM, Lee JW, Eom SH, Hwang IK, Lee
CY. Protective effect of steamed American ginseng
S198 J Med Assoc Thai Vol. 100 Suppl. 5 2017
(Panax quinquefolius L.) on V79-4 cells induced
by oxidative stress. J Ethnopharmacol 2007; 111:
443-50.
13. Potent antioxidative and UVB protective effect of
water extract of Eclipta prostrata L. Sci World J
2014; [8 pages]. Article ID 759039. http://dx.doi.
org/10.1155/2014/759039.
14. Pukumpuang W, Chansakaow S, Tragoolpua Y.
Antioxidant activity, phenolic compound content
and phytochemical constituents of Eclipta
prostrata Linn. Chiang Mai J Sci 2014; 41: 568-76.
15. Datta K, Singh AT, Mukherjee A, Bhat B,
Ramesh B, Burman AC. Eclipta alba extract with
potential for hair growth promoting activity. J
Ethnopharmacol 2009; 124: 450-6.
J Med Assoc Thai Vol. 100 Suppl. 5 2017 S199
⌦ ⌫
⌫⌫
⌫
⌫⌫⌫ ⌫⌫
⌦
⌦⌫⌫
⌫ ⌫ ⌫
⌦ ⌫
⌫⌫
⌫ ⌫ ⌫
⌦⌫
⌫
⌦ ⌫⌫⌫⌫ ⌦⌫ μ
⌫ ⌫⌫⌫
⌫⌫⌫⌫ μ
⌫ ⌫ μ ⌫⌫⌫
⌧ ⌫
⌫ μ ⌫⌫⌫ ⌫ μ
⌦⌫⌫⌫⌫
⌫ ⌫⌫⌫⌫⌫⌫⌫⌫
⌦⌫⌫⌫⌫⌫
